Field of Invention
The present invention relates to the field of nucleic acid amplification, particularly to the use of a polymerase chain reaction to amplify nucleic acids. The invention provides methods and kits which can be used to amplify nucleic acids by combining a solid support such as an FTA™ paper with PCR reagents for one step amplification of nucleic acid samples. The invention has applications in the long term storage and easy processing of nucleic acids and is particularly useful in genotyping, diagnostics and forensics.
Background of the Invention
The polymerase chain reaction (PCR) is a common tool used in molecular biology for amplifying nucleic acids. U.S. Pat. No. 4,683,202 (Mullis, Cetus Corporation) describes a process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof.
Long-term storage, transport and archiving of nucleic acids on filter paper or chemically modified matrices is a well-known technique for preserving genetic material before the DNA or RNA is extracted and isolated in a form for use in genetic analysis such as PCR. Thus, EP 1563091 (Smith et al, Whatman) relates to methods for storing nucleic acids from samples such as cells or cell lysates. The nucleic acid is isolated and stored for extended periods of time, at room temperature and humidity, on a wide variety of filters and other types of solid support or solid phase media. Moreover, the document describes methods for storing nucleic acid-containing samples on a wide range of solid support matrices in tubes, columns, or multiwell plates.
WO 90/03959 (Burgoyne) describes a cellulose-based solid support for the storage of DNA, including blood DNA, comprising a solid matrix having a compound or composition which protects against degradation of DNA incorporated into or absorbed on the matrix. This document also discloses methods for storage of DNA using the solid medium, and for recovery of or in situ use of DNA.
U.S. Pat. No. 5,496,562 (Burgoyne) describes a cellulose-based solid medium and method for DNA storage. Method for storage and transport of DNA on the solid medium, as well as methods which involve either (a) the recovery of the DNA from the solid medium or (b) the use of the DNA in situ on the solid medium (for example, DNA sequence amplification by PCR) are disclosed. Unfortunately, the methods described only incorporates a surfactant or detergent on the surface of the solid medium and therefore suffer from the disadvantage that they require a separate step for the removal of the detergent before PCR is performed.
EP 2290099 B1 (Qiagen) describes again a method for processing and amplifying DNA. The method includes the steps of contacting the sample containing DNA to a solid support wherein a lysis reagent is bound to the solid support. The DNA is subsequently treated with a DNA purifying reagent and is purified. The application does not include a sequestrant on the solid support and requires a separate step for the removal of the lysis reagent and purification of the DNA before amplification.
WO 96/39813 (Burgoyne) describes a solid medium for storing a sample of genetic material and subsequent analysis; the solid medium comprising a protein denaturing agent and a chelating agent. The method described is for chelating agents which are any compound capable of complexing multivalent ions including Group II and Group III multivalent metal ions and transition metal ions. The invention does not specifically mention cyclodextrin as a chelating agent, nor does it suggest the PCR analysis could be performed in a single step.
U.S. Pat. No. 5,705,345 (Lundin et al.) describes a method of nucleic acid preparation whereby the sample containing cells is lysed to release nucleic acid and the sample is treated with cyclodextrin to neutralize the extractant. The advantage of this system is that conventional detergent removal requires a separation step however with the addition of cyclodextrin to neutralize the detergent it would remove the separation step needed and reduce chance of contamination.
GB 2346370 (Cambridge Molecular Technologies Ltd) describes applying a sample comprising cells containing nucleic acid to a filter, the cells are retained by the filter and contaminants are not. The cells are lysed on the filter and retained alongside the nucleic acid. Subsequent steps filter out the cell lysate while retaining the nucleic acid.
WO 96/18731 (Deggerdal) describes a method of isolating nucleic acid whereby the sample is bound to a solid support and sample is contacted with a detergent and subsequent steps performed to isolate the nucleic acid.
WO 00/53807 (Smith, Whatman) describes a medium for the storage and lysis of samples containing genetic material which can be eluted and analyzed. The medium is coated with a lysis reagent. In addition the medium could be coated with a weak base, a chelating agent, a surfactant and optionally uric acid.
WO 99/38962 (Health, Gentra Systems Inc.) describes a solid support with a bound lysis reagent. The lysis reagent can comprise of a detergent, a chelating agent, water and optionally an RNA digesting enzyme. The solid support does not contain cyclodextrin and requires further steps for purification of the nucleic acid for amplification analysis.
Current methods for DNA amplification involve a DNA purification procedure which often involves several steps which increases the chance of contamination. This is a tedious process and prior art methods have a number of clear disadvantages in terms of cost, complexity and in particular, user time. For example, column-based nucleic acid purification is a typical solid phase extraction method to purify nucleic acids. This method relies on the nucleic acid binding through adsorption to silica or other support depending on the pH and the salt content of the buffer. Examples of suitable buffers include Tris-EDTA (TE) buffer or Phosphate buffer (used in DNA microarray experiments due to the reactive amines). The purification of nucleic acids on such spin columns includes a number of complex and tedious steps. Nucleic acid purification on spin columns typically involves three time-consuming and complex steps/stages: the sample containing nucleic acid is added to the column and the nucleic acid binds due to the lower pH (relative to the silanol groups on the column) and salt concentration of the binding solution, which may contain buffer, a denaturing agent (such as guanidine hydrochloride), Triton X-100, isopropanol and a pH indicator;
the column is washed with 5 mM KPO4 pH 8.0 or similar, 80% EtOH); and
the column is eluted with buffer or water.
Alternative methods involve the binding of nucleic acids in the presence of chaotropic salts such that DNA binds to silica or glass particles or glass beads. This property was used to purify nucleic acid using glass powder or silica beads under alkaline conditions. Typical chaotropic salts include guanidinium thiocyanate or guanidinium hydrochloride and recently glass beads have been substituted with glass containing minicolumns.
The best defense against PCR amplification failure in forensics applications is to combine sound sample handling and processing techniques with extraction systems proven to efficiently purify DNA.
Santos C. R. et al., (Brazilian Journal of Microbiology, 2012, 43, 389-392) describes a method of skipping the elution step prior to PCR amplification of nucleic acid and adding the punches directly into the PCR mix. The PCR amplification was performed for the detection of HPV-DNA and was more efficient than the standard FTA elute card protocol of eluting the nucleic acid prior to amplification. However this method only used qualitative PCR to measure the presence of HPV.
Nozawa N. et al., (Journal of Clinical Microbiology, 2007, 45, 1305-1307) describes a method of using real time PCR for the detection of cytomegalovirus (CMV). The method described involved the use of filter paper with purified CMV and was added directly to the PCR mix. The paper noted that only instruments with a photo-multiplier-tube scanning system could be used for real time PCR assays with filter disks. The paper suggests that the filter paper would adversely affect instruments using a charge-coupled device camera and therefore teaches away from the use of filter papers in real time PCR machines such as an ABI7700 machine.
Qiagen Sample & Assay Technologies Newsletter (March 2010, 15) describes the effects of a low A260/A230 ratio in RNA preparations on downstream PCR processing. The newsletter notes that increased absorbance at 230 nm in RNA samples is quite often due to contamination with guanidine thiocynate (a component of FTA elute cards and is used in RNA purification procedures). The experiments demonstrate A260/A230 ratio of an RNA sample is lower when guanidine thiocyanate is present, however guanidine thiocyanate concentrations up to 100 mM in an RNA sample did not affect the reliability of real-time PCR.
Typically the purification steps involved in the standard FTA elute card protocol can be cumbersome and purification can lead to a loss in DNA quality. There is therefore a need for an improved and simplified process for amplifying, quantifying and or profiling nucleic acid, which removes the need for a purification step. The present invention addresses this problem and provides methods and kits which can be used for single step amplification of nucleic acid from solid supports, particularly cellulose-derived supports.